Method for the production of microbubble-type ultrasonic contrast agent by surfactant

ABSTRACT

An imaging agent for ultrasound to be administered into a vein is provided. More specifically, an imaging agent for ultrasound containing microbubbles, which has a long-life and is stable within a human body even after is has passed through a lung, and a method for producing the same, are provided. 
     A method for producing an imaging agent for ultrasound comprises a mixing step wherein a mixture of water, Sodium Stearate, Saponin and CaCl 2  and gas are mixed by a homogenizer for forming bubbles, and a separation step wherein bubbles having a desired size are separated according to their buoyancy after the mixing step. The mixing step further includes two stages, i.e., a low speed mixing stage and a high speed mixing stage. In the low speed mixing stage, a shaft of the homogenizer is immersed into a solution containing a preset amount of Sodium Stearate, Saponin, and CaCl 2 , which is mixed at a low rotation speed for a given period of time and is then left for a given period. Then, such a process is repeated. In the high speed mixing stage, said solution is mixed for a given period at a high rotation speed. After this 2-stage mixing step, the separation is performed by using a burette, thereby obtaining microbubbles of a desired size.

This is a division of application Ser. No. 08/509,942 filed Aug. 1,1995, U.S. Pat. No. 5,876,647. The entire disclosure of the priorapplication(s) is hereby incorporated by reference herein in itsentirety.

FIELD OF INVENTION

This invention relates to methods for the production of long-lifestable, uniformly small size microbubbles which has the potential to beuseful as a contrast agent in ultrasonic imaging.

BACKGROUND OF INVENTION

Various technologies exist in which parts of an animal or human body maybe imaged so as to aid in diagnosis and therapy of medical disorders.Some of these existing techniques are described in this section.

X-ray is the most well-known imaging techniques to visualize skeletaland other internal structures within animals and humans. However, anumber of problems associates with the use of X-rays. Firstly, X-ray isnot a safe diagnostic method in visualizing some parts of the humanbody, the use of X-ray for some of the organs and blood vessels isunsatisfactory. In addition, X-ray is dangerous if the amount ofexposure is excessive; further, all X-ray radiation absorbed over alifetime is cumulative.

Another technique, radio-nuclide imaging involves the injection ofradioactive substances, such as thallium into the bloodstream. Thistechnique require the use of very expensive and sophisticated machinery.Further, radionuclide imaging produces images of only a limited numberof view of the heart and those images may not be of exceptional clarity.Finally, this type of radiation is cumulative over a lifetime and thisis hazardous.

Ultrasound imaging techniques are safe, cheap, relatively easy tooperate and the image produced is in real-time. Therefore, ultrasound iswidely used in diagnostic imaging nowadays. The basis for ultrasoundimaging is that ultrasonic waves are send into human body by anultrasound probe and the waves reflected from the tissues are detectedby the same probe, the received signal is then processed to produceimages on a monitor by a ultrasonic diagnostic instrument. According todifferent acoustic properties of different tissues in the human body,the diagnosis of diseased tissues can be distinguished from normaltissues quickly by observing the real-time image produced on themonitor. In addition to diagnostic application, the visualization ofblood flow is also a common application of ultrasound in medicalimaging, by monitoring the change in frequency of ultrasonic waves sentto blood vessels and the reflected waves. The blood flow velocity can becalculated.

However, in these field of application, the lack of clarity ofultrasound is still a very big problem to be solved.

Since early ultrasound techniques suffered from a lack of clarity,extensive efforts were undertaken to improve the ultrasonic equipment.To deal with this problem, it is valuable to mention that whenultrasonic energy is directed through substances, changes in thesubstances' acoustic properties will be most prominent at the interfaceof different media (i.e. solids, liquids and gases). As a consequence,when ultrasound energy is directed through various media, the reflectionof ultrasound at the interface of different media will be the strongestand can be detected most easily. That is the basic principle for themaking of ultrasonic contrast agents.

Contrast agents were introduced into the bloodstream in an effort toobtain enhanced images. The maximum ultrasonic reflection that we canobtain is that from the interface between liquid and gaseous media. Thatis why many of these contrast agents were liquids containingmicrobubbles of gas. These contrast agents themselves are intense soundwave reflectors because of acoustic differences between the liquids andthe gas microbubbles enclosed therein. When the contrast agents areinjected into and perfuse the microvasculative of tissue, clearer imagesof such tissue may be produced. However, not withstanding the use ofsuch contrast agents, the image produced, for example, of the myocardialtissue, is of relatively poor quality, is highly variable and is notquantifiable due to the variable size and persistence associated withprior art microbubbles.

In the recent years, much effort was made to manufacture microbubbletype contrast agents which includes Albunex® by Widder et al andLipid-Coated Microbubbles (LCM) by D'Arrigo. For the commerciallyexisting ultrasonic contrast agent Albunex®, although has a reportedmean diameter under 6 μm, up to 0.5% of the microbubbles, or up to2,000,000 microbubbles/ml are over 9 μm in diameter in the fullypurified product. According to J. Ophir, the microbubbles which areabove 3 to 5 μm will not pass through the lung capillaries. Therefore,the microbubbles cannot reach the organs to provide diagnosticproperties. In this sense, microbubbles sized below 5 μm should be madein order to overcome this problem. Overcoming the problem of size,D'Arrigo successfully made Lipid-Coated Microbubbles (LCM) of which 99%of the microbubbles are sized below 4.5 μm in diameter by the use ofsurfactants. However, the number of microbubbles made in D'Arrigo'sinvention is much less than that of Albunex® which is in the order of1×10⁸. The number of microbubbles made by D'Arrigo is in the order of1×10⁶. For the best imaging properties to occur, it is desirable to havehigher concentration of microbubbles. Furthermore, there is not yet amethod or a effective way to control the size of the microbubblesproduced which may suit different conditions for other commercial uses.

SUMMARY OF INVENTION

This invention is directed to an improvement associated with producingmicrobubble type ultrasonic contrast agent by which large concentrationof small and uniform size of microbubbles are produced in water base. Asecond embodiment is directed to the method to control the size ofmicrobubbles produced in order to suit different situations.

The present invention is directed to methods to produce largeconcentration, room temperature stable microbubble type contrast agentswhich involves procedures that are capable of producing microbubbleswith the size range from less than 1 μm to 200 μm, and then microbubblesof different sizes can be collected easily within this size range.

The ultrasonic contrast agents in the present invention are microbubblesof the following properties

(i) ultrasonically echogenic (i.e. capable of reflecting sound waves).

(ii) uniform size and small enough to pass through lung capillarysystem, thereby producing enhanced image of various kinds of tissues andorgans and permitting differentiation between well-perfused andpoorly-perfused tissue.

(iii) quantifiably reproducible.

(iv) long-life for storage in vitro.

The basic components of this invention comprises

(1) Homogenizer of which

(a) the shaft can rotate at a speed (from 0 to above 20000 rpm) and itcan rapidly introduce bubbles into the solution in which the shaft isimmersed in the liquid.

(b) has hole on the shaft which is capable of sucking air or gases fromthe outside environment. After the air is sucked from the outside, bythe high-speed rotating motion, the air is rapidly introduced into thesolution.

(c) the basic function is to make particle smaller by mechanical means.In this invention, a new function of the homogenizer is introduced thatthe homogenizer can be used to produce microbubbles of smaller sizerange from 0.5 μm to 5 μm.

(2) a member chosen from the group of surfactants consisting of SodiumSalt of saturated carboxylic acids containing from about 10 to about 18carbon atoms. Examples include Sodium Stearate, Sodium Mystritate,Sodium Palmitate, Sodium Laurate, Sodium Oleate . . . etc.

(3) Another material chosen from the group of surfactants which iscurrently used as producing bubbles which includes

(i) Saponin

(ii) Stearic acid

(iii) Phloxine

(iv) Crystal Violet

(v) Polyvinyl alcohol

(vi) Sodium Laurate

which is capable of stabilizing microbubbles produced from surfactantdescribed above in (2) temporarily and the other function is to slowdown the rapid reaction between surfactant mentioned (2) and thestabilizer that will be described immediately in point (4).

(4) a member chosen from the group of reagents which can change thenature of the surface of the microbubbles produced from the surfactantsmentioned in point 2 above. These include the salts when dissolved inwater, the cation come out will have a reaction which the group (2) and(3) mentioned above. Examples of these are Calcium Chloride andMagnesium Chloride.

(5) a separation procedure to enable us to collect different range ofsize of bubbles according to our interest.

Firstly a mixture is made by admixing solid selected from group (2) and(3) described above.

Said mixture from group (2) and (3) in the said ratio 1.0:0.1-1.0:2.0.

Said mixture is put into pure water to make a dilute solution. After thesolution is made, to ensure that the surfactants are fully dispersed inthe pure water and to produce a concentrated emulsion of microbubbles, astepwise-homogenizing technique is used. That is, first the solution ishomogenzied at slower homogenizer speed for a short time, and repeat theabove procedure once. Then a faster homogenizer speed and longer time ofhomogenizing is used to produce an emulsion which contains large amountof microbubbles. The main use of chemicals mentioned in group (3) inthis invention is to stabilize the microbubbles produced from group (2)for a short period of time before another stabilizer is added tostabilize the microbubbles produced. By controlling the procedure ofhomogenizing, such as homogenizing speed and time, we can control thesize and amount of bubbles produced quantitatively.

Then the emulsion is put into a burette. Burette is a glass tube with astopcock at the bottom. After the emulsion is put into the tube, becauseof the faster rising time of larger bubbles, different sizes of bubblescan be obtained at different levels of the burette after a period oftime.

By collecting the microbubbles at the bottom of the burette. We canobtain the microbubble of the smallest size which ranges from 0.5 μm to5 μm.

After microbubbles are collected a small amount of 0.2-30% of solutionfrom group (4) is added into the bubble solution to change the nature ofthe coating produced by the surfactants. Thereby, stabilizing the bubbleproduced.

According to different purpose of usage, different sizes of bubbles canbe obtained by carefully controlling the homogenizing speed and time,and the collection time and level. The microbubbles exists for at least6 months after they are produced.

In this invention, the mean bubble diameter produced can be smaller than0.5 μm and 99% of microbubble diameter is less than 3 μm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the graphically illustration of the method of production ofmicrobubbles.

FIG. 2 is a histogram graphically illustrating a typical particle sizedistribution of stable microbubbles obtained in accordance with themethod of the present invention, as determined by Coulter multisizer.The mean particle diameter is measured to be 0.910 μm. 99% ofmicrobubbles less than 1.346 μm.

FIG. 3 is a histogram graphically illustrating the particle sizedistribution of stable microbubbles of which the size is greater thanthat in FIG. 1. The mean particle diameter is 1.030 μm and 99% ofparticle size is less than 3.345 μm. The different in mean size isobtained intentionally by the method described in this invention.

FIG. 4 is a histogram graphically illustrating the particle sizedistribution of microbubbles prepared in this invention according toexample 4 mentioned below.

FIGS. 5A and 5B are photographs showing the microbubbles observed by 600times microscopy. FIG. 5B: the photograph of the bubble sample mentionedin FIG. 1 shown above. 5A: the photograph of the bubble simple mentionedin FIG. 2 shown above.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to producing microbubble type contrast agentwhich is

(i) biocompatiable or biodegradable.

(ii) small enough to pass through lungs capillary beds.

(iii) ultrasonically echogenic.

(iv) stable for storage at room condition.

(v) quantitatively reproducible.

In this invention, a method is introduced to produce microbubbles ofsize range as small as from under 0.5 μm to 5 μm which can exist underroom temperature storage for at least 6 months. The basic components inthis invention include

(1) Homogenizer of which

(a) the shaft can rotate at a speed (from 0 to above 20000 rpm) and itcan rapidly introduce bubbles into the solution in which the shaft isimmersed.

(b) has a hole on the shaft which is capable of sucking air or gasesfrom the outside environment. After the air is sucked from the outside,by the high-speed rotating, motion, the air is rapidly introduced intothe solution.

(c) the basic function is to make particles smaller by mechanicalmeaning.

In this invention, a new function of the homogenizer is introduced thatthe homogenizer can be used to produce microbubbles of a smaller sizerange of from less than 0.5 μm to 5 μm.

Homogenizer is commercially available machine that can be available fromNition, Kinematica, Hitachi, Homogenizer Polytron, or IKA Ultrat-Turraxor other companies.

Also there are other kinds of machines which are commercially availablethat can be used to make microbubbles, such is Vibro Mixer, Poem Labo ofReica.

Also bubbles can also be produced by other kinds of equipment such as anatomizer.

(2) a member chosen from the group of surfactants consisting of SodiumSalt of saturated carboxylic acids containing from about 10 to about 18carbon atoms. Examples include Sodium Stearate, Sodium Laurate, SodiumOleate . . . etc.

(3) Another material chosen from the group of surfactants which iscurrently used as producing bubbles which includes

(i) Saponin

(ii) Stearic acid

(iii) Phloxine

(iv) Crystal Violet

(v) Polyvinyl alcohol

(vi) Sodium Laurate

which is capable of producing and stabilizing microbubbles produced fromthe surfactant described above in (2) temporarily and the other functionis to slow down the rapid reaction between the surfactant mentionedabove in (2) and the stabilizer that will be described immediately in(4) below.

(4) a member chosen from the group of reagents which can change thenature of the surface of the microbubbles produced from the procedurecombining (1) (2) and (3) above. These are the salts that when dissolvedin water, a cation comes out that will have a reaction with the group(2) and (3) mentioned above. Examples of these arc Calcium Chloride andMagnesium Chloride.

(5) a separation procedure to enable us to collect different range ofsize of bubbles according to our interest.

Firstly a mixture is made by admixing solid selected from group (2) and(3) described above.

Said mixture from group (2) and (3) in the said ratio 1:0.1-1:2.0.

The procedure in producing microbubbles is first taking small amount ofsurfactant from components (2) and (3) described above and put it intopure water making a 0.1% solution. To distribute the solid surfactantwell in the solution and produce concentrated bubble solution, astepwise homogenizing technique is used. The stepwise homogenizingtechnique includes three procedures. Firstly after the mixture producedabove is put into the pure water, the solution is homogenized at a lowhomogenizing speed for a short time and the process is repeated once. Awhite emulsion is obtained. Then a higher homogenizing speed is thenapplied to the emulsion for a longer time. An emulsion concentrated withbubbles ranging from less than 1 μm up to 70 μm is produced.

By process of separation of bubbles by the use of burette (which is along glass tube with a stopcock at the bottom), small size bubbles canbe obtained. After the concentrated bubble solution is produced, theemulsion is put into a burette. Since larger size bubbles will rise upthe burette faster than smaller bubbles. So that after storing thebubbles in the burette for a period of time, the microbubbles at thebottom of the burette will be only small size bubbles. Only bubbles atthe bottom of the burette are collected. The storage in burette can bein 4° C. or 24° C. or at room condition. The microbubbles that arelarger than required are discarded.

After the microbubble emulsion is collected from the burette, anothermaterial is used which can change the chemical nature of the coating ofmicrobubbles is added to the microbubble emulsion. Small amount of0.2-30% solution of the chemical described in group (4) is added to theemulsion collected. These chemicals can be said to be a stabilizer ofmicrobubbles produced. By these processes, stabilized small microbubbleswhich are of size from less than 1 to 5 μm are formed. The bubbles canexist for at least 9 months after production.

Particle size analysis is determined by electroimpedance-sensedvolumetric sizing multisizer. In this invention, microbubbles of numberin the order of 1×10⁹ per milliliter with the mean size less than 1 μmis produced. In addition, 99% of the microbubbles produced is less than3 μm in diameter.

Also by this method of collecting of microbubbles, we can makemicrobubbles of the range from less than 1 μm to 4.5 μm and from lessthan 1 μm to 16 μm in diameter. By controlling the homogenizing time andspeed and the materials used, we also can control microbubbles of thesize range between 0.1 and 200 μm in diameter.

EXAMPLE 1

This example illustrates the preparation of surfactant mixture. A drypowdery surfactant mixture was prepared in accordance with the presentinvention by admixing Sodium Stearate and Saponin in the ratio1:0.1-1:1.

EXAMPLE 2

This example illustrates the method of preparation of stablegas-in-liquid emulsion. 80 mg of surfactant mixture as produced fromexample 1 is used. The solution is first homogenized at a 5000 rpm for 1minutes and wait for 2 minutes for dispersion and the process isrepeated once. Then the solution is homogenized at a 10000 rpm toproduce large amount of bubbles.

Separation Method

The emulsion from example 1 is put into a 25 ml burette. Then afterputting into the burette into a refrigerator at 4° C. for 1 hour, 2.5 mlof bubble solution is taken from the bottom of the burette.

Stabilization

Then 200 μl of 3% CaCl₂ solution is put into the 2.5 ml of microbubblesolution. The mean size of microbubbles is measured to be 0.971 μm andthe bubble number is measured to 1.3668×10⁹ per millimeter. 99% ofmicrobubbles is less than the size 2.2708 μm.

EXAMPLE 3

This example illustrates the significance of using the burette as themethod to collect different mean size of microbubbles. The microbubblesis collected from the level 2.5 ml to 5 ml from the burette. The meansize is measured to be 1.0122 μm and the bubble number is measured to be1.697×10⁹ per millimeter. 99% of bubble is less than size 3.578 μm indiameter.

EXAMPLE 4

This example illustrates the effect of different homogenizer speed andhomogenizing time on the size of microbubbles produced. The surfactantmixture is produced as in example 1 and example 2. 80 mg of surfactantmixture is put into 80 ml of water and the solution is homogenized firstat a speed of 5000 rpm for 30 seconds and then wait for 1 and a halfminutes and then the solution is homogenized as a 7500 for 10 seconds.15 μm to 65 μm of microbubbles is produced. Also by further decreasingthe homogenizer speed, the size of microbubbles produced can be up to200 μm.

EXAMPLE 5

This example illustrate about the effect of toxicity of microbubbles.Bubbles are manufactured as illustrated in example 1 and example 2. 18Wistar rats with 9 male and 9 female rats ranging from 300 to 400 g aredivided into three groups to receive three kinds of dose including

(i) 1 ml microbubble solution per 250 g weight of rat.

(ii) 0.5 ml microbubble solution per 250 g weight of rat.

(iii) 0.2 ml microbubble solution per 250 g weight of rat.

These three kinds of dose are injected into the 18 rats everyday for 14days. No rat died after injection for 14 days, and there is noobservable abnormality in the behavior of all the rats. This phenomenonillustrates that the microbubbles have no fatal toxicity.

What is claimed is:
 1. A method for preparing an imaging agent forultrasound comprising the steps of:A) obtaining an aqueous surfactantmixture for producing a stable microbubble solution containing:(a) atleast one first surfactant selected from the group consisting of sodiumlaurate, sodium myristate, sodium palmitate, sodium stearate andmixtures thereof, and (b) at least one second surfactant selected fromthe group consisting of saponin, phloxine, Crystal Violet and polyvinylalcohol; B) forming bubbles in the aqueous surfactant mixture bysubjecting the aqueous surfactant mixture to a high speed mixing using amachine having a shaft rotating at a high rotational speed from 5,000 toover 20,000 rpm, and at the same time, introducing gas into the aqueoussurfactant mixture, thereby producing a microbubble solution comprisinga dispersion of microbubbles of sizes from under 1 μm to over 80 μm; C)pouring the microbubble solution into a tube with a stopcock at thebottom and leaving the tube standing a period of time; D) collecting themicrobubble solution from the bottom of the tube; and E) stabilizing themicrobubble solution by adding a stabilizing solution containing calciumchloride or magnesium chloride to the collected microbubble solution inorder to change the nature of the surface of the microbubbles.
 2. Themethod according to claim 1, wherein the machine used is a homogenizeror a Vibro Mixer.
 3. The method according to claim 1, wherein theaqueous surfactant mixture has a weight ratio of the first surfactant tothe second surfactant in the range of 1:0.05-1:2.
 4. The methodaccording to claim 1, wherein the aqueous surfactant mixture has aweight ratio of the first surfactant to the second surfactant equal to 1to
 1. 5. The method according to claim 1, wherein the aqueous surfactantmixture is formed by dissolving 80 mg of the first and secondsurfactants in 80 ml of water.
 6. The method according to claim 1,wherein the bubble forming step B comprises a low speed mixing of theaqueous surfactant mixture prior to subjecting the aqueous surfactantmixture to a high speed of rotation to produce high concentration ofmicrobubble solution.
 7. The method according to claim 6, furthercomprising the step of subjecting the aqueous surfactant mixture to asecond low speed mixing process before the high speed mixing process. 8.The method of claim 1, in which the microbubble solution produced isvaried in accordance with homogenizing speed and time.
 9. The method ofclaim 1, in which different range of size microbubble is collected. 10.The method according to claim 1, wherein said stabilized microbubblesolution is formed with substantially 99% of the microbubbles havingdiameters less than 3 μm.
 11. The method according to claim 1, whereinsaid stabilized microbubble solution occurs when the concentration ofthe microbubble is more than 1×10⁹ microspheres per milliliter.
 12. Themethod according to claim 1, wherein the stabilized microbubble solutionproduced is stable for over 9 months at room condition.
 13. The methodaccording to claim 1, wherein the first surfactant consists of sodiumstearate mixed with at least one of sodium laurate, sodium myristate andsodium palmitate.
 14. The method according to claim 13, wherein thesecond surfactant is saponin.
 15. The method according to claim 1,wherein the first surfactant is sodium laurate, sodium myristate ormixtures thereof.
 16. The method according to claim 1, wherein thesecond surfactant is one or more of phloxine, Crystal Violet orpolyvinyl alcohol.